title: Sample 8
description: Protocols: The procedure for collection and incubation of the porcine CL was previously described [20]. In the laboratory, the CL was separated from the ovary, cleaned and placed on ice in a sterile Petri dish. CLs were cut into small pieces (100 ± 10 mg, in duplicate from each experimental replicate). Each tissue explant was placed in M199 medium (Sigma Aldrich, St. Louis, MO, USA) supplemented with 0.1% BSA fraction V (Roth, Germany) and antibiotics. The explants were pre-incubated for 2 h in a water bath at 37°C in an atmosphere of 95% O2 and 5% CO2. Then, the explants were treated with LPS (100 ng/ml, from Escherichia coli) for 24 h. Explants not treated with LPS were considered as controls. The medium was removed, and the explants were incubated for 6 h with LPS alone or in combination with PPAR β/δ ligands: GW0724 (agonist; 1 μmol/L or 10 μmol/L, Cayman Chemical Company) or GSK3787 (antagonist; 25 μmol/L, Cayman Chemical Company). Controls also contained dimethyl sulfoxide (DMSO, solvent for the tested PPAR ligands). After the incubation, tissue explants were frozen at -80°C until further analysis. Total RNA from 16 samples was isolated using the \RNeasy Mini Kit\ (Qiagen, Germany) according to the manufacturer's protocol. The Tecan Infinite M200 plate reader (Tecan Group Ltd., Switzerland) and Agilent Bioanalyzer 2100 (Agilent Technology, USA) were used to evaluate total RNA quantity and quality. The samples with RNA Integrity Number (RIN) > 7 were selected for the next analyses. The poly(A) RNA-sequencing library was prepared according to the Illumina TruSeq Stranded mRNA Sample Preparation Protocol. Two rounds of purification were performed using oligo(dT) magnetic beads to purify the poly(A) tailed mRNA. Subsequently, the poly(A) RNA was fragmented at high temperature using a divalent cation buffer, and poly(dT) oligonucleotides were used to transcribe the RNA into cDNA. Subsequently, the cDNA was subjected to 3' tail adenylation and adapter ligation. Reverse transcription during library construction was strand-specific
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2022-09-01
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2022-09-01
attributes.tag: External Id
attributes.value: SAMEA110463622
attributes.tag: INSDC center alias
attributes.value: Department of Animal Anatomy and Physiology, Faculty of Biology and Biotechnology, University of Warmia and Mazury
attributes.tag: INSDC center name
attributes.value: Department of Animal Anatomy and Physiology, Faculty of Biology and Biotechnology, University of Warmia and Mazury
attributes.tag: INSDC first public
attributes.value: 2022-09-01T00:14:24Z
attributes.tag: INSDC last update
attributes.value: 2022-09-01T00:14:24Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-12027:Sample 8
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: pig
attributes.tag: growth condition
attributes.value: explant culture
attributes.tag: organism part
attributes.value: ovary
attributes.tag: sample name
attributes.value: E-MTAB-12027:Sample 8
attributes.tag: sampling site
attributes.value: corpus luteum
attributes.tag: sampling time point
attributes.value: mid-luteal phase